Transposon mutagenesis of baculoviruses provides an ideal experimental system for analysis of the movement of a unique family of mobile element identified from lepidopteran genomes. Members of this family of short-inverted-repeat elements are characterized by their extreme specificity for TTAA target sites. This report describes the analysis of excision events for two representatives of this family, tagalong (formerly TFP3) and piggyBac (formerly IFP2). These elements were tagged with a polyhedrin/lacZ reporter gene and inserted back into the virus genome either by homologous recombination or by transposition. Revertants were selected based on a white plaque phenotype. Both elements excise in a precise fashion from their positions in the baculovirus genome in either TN-368 cells or IPLB-SF21 AE cells. The precise excision of these elements in infected IPLB-SF21 AE cells occurs in the absence of either tagalong or piggyBac element encoded functions. The common characteristics of both insertion and excision for these elements provides further validation for their inclusion in a single family of unique transposons.

The piggyBac Lepidopteran transposable element moves from the cellular genome into infecting baculovirus genomes during passage of the virus in cultured TN-368 cells. We have constructed genetically tagged piggyBac elements that permit analysis of excision when transiently introduced on plasmids into the piggyBac-deficient Spodoptera frugiperda IPLB-SF21AE cell line. Precise excision of the element from these plasmids occurs at a higher frequency in the presence of a helper plasmid that presumably supplies the piggyBac transposase. The results suggest that the piggyBac transposon encodes a protein that functions to facilitate not only insertion, but precise excision as well. This is the first demonstration of piggyBac mobility from plasmid sources in uninfected Lepidopteran cells.

The terminal DNA sequence requirements for piggyBac transposable element excision were explored using a plasmid-based assay in transfected, cultured insect cells. A donor plasmid containing duplicate 3' piggyBac terminal inverted repeats was constructed that allowed individual nucleotides or groups of nucleotides within one of the 3' repeats to be mutated. The relative extent of excision using the mutated end versus the wild-type end was then assayed. Removal of even one of the terminal 3' G nucleotides from the piggyBac inverted repeat, or removal of the dinucleotide AA from the flanking TTAA target site prevents excision of piggyBac at the mutated terminus. Incorporation of an asymmetric TTAC target site at the 3' end does not prevent excision from the mutated end. Thus, both piggyBac DNA and flanking host DNA appear to play crucial roles in the excision process.

The IFP2 element is a unique Lepidopteran transposon that has been associated with spontaneous Baculovirus mutants isolated following passage of the virus in the TN-368 cell line. Independent genomic representatives of IFP2 from TN-368 cells show little sequence divergence, suggesting that IFP2 was recently introduced into this genome and is highly stable. IFP2 is inserted within AT-rich regions of the TN-368 genome and targets TTAA sites. The specificity for TTAA target sites during transposition is not limited to the movement of IFP2 during an active Baculovirus infection, but is a property of its movement in uninfected cells as well. The exact origin of IFP2 remains obscure since it is found in two independently established Trichoplusia ni cell lines but not in three others, and we have not yet identified any IFP2 sequences in either field collected larvae or laboratory colonies.

The transposons piggyBac and tagalong are Lepidopteran transposons that exhibit extreme site-specificity for the tetranucleotide TTAA upon insertion and excise in a characteristic precise fashion, regenerating a single TTAA target site. The precise excision of both piggyBac and tagalong can occur in the absence of factors encoded by either transposon, possibly through the recruitment of host proteins and/or cross-mobilizing transposons. In this report, we utilize mobility shift assays and exonuclease III protection analyses to identify DNA binding activities from IPLB-SF21AE and TN-368 cells that are specific for piggyBac and tagalong-terminal repeats.